Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. Hypoxia/contraction in muscle also stimulates glucose transport activity and this effect is additive to that of insulin. However, quantification of these processes by classical subcellular fractionation techniques has been very difficult because of the contaminating microfibrillar protein and dynamic studies at the molecular level have been almost impossible. Thus, we have prepared a muscle-specific transgenic mouse model in which HA-GLUT4-GFP is expressed under the control of the MCK promoter. Western blotting and confocal microscopy confirm the specificity and levels of HA-GLUT4-GFP expression in intact muscles, and isolated FDB muscle fibers and cardiomyocytes (Fazakerley et al.). Confocal microscopy further demonstrates that HA-GLUT4-GFP is translocated to PM and the t-tubules of isolated FDB fibers to the same degree after insulin and hypoxia, and that these effects are additive. However, TIRFM reveals a remarkable difference in dynamics between adipose and muscle cells: In muscle, in either isolated FDB fibers or soleus muscles in vitro, or in gastrocnemius muscles in living animals, in the non-stimulated state almost all GLUT4 are found in PM clusters and only very few are mobile. Furthermore, stimulation of 2-DOG transport in muscle by insulin is typically 2-3-fold, the same magnitude as that of the stimulation by insulin of fusion events at the GLUT4 clusters in adipose cells (2-fold, see poster by Stenkula et al.). Finally, the Ploug laboratory (Lauritzen et al.) has reported that in skeletal muscle, insulin reversibly stimulates local depletion of GLUT4 storage vesicles at sarcolemma and t-tubules rather than inducing movement of intact storage vesicles. These data together strongly suggest that GLUT4 clusters (called hubs in adipose cells) are the major site of glucose transport regulation in muscle, and that GLUT4 are released from these hubs in response to stimuli, and recycle through these hubs during, and reaccumulate in these hubs after, stimulation. A new mouse model has been developed to study the localisation and trafficking of the glucose transporter GLUT4 in muscle. The mouse line has specific expression of a GFP and HA-epitope-tagged version of GLUT4 under the control of a muscle-specific promoter. The exofacial HA-tag has enabled fluorescent labelling of only the GLUT4 exposed at the external surface. A distinction between sarcolemma labelling and transverse-tubule labelling has also been possible because the former compartment is much more accessible to intact anti-HA antibody. By contrast, the Fab fragment of the anti-HA antibody could readily detect GLUT4 at the surface of both the sarcolemma and transverse tubules. Here, we have used this mouse model to examine the route taken by cardiomyocyte GLUT4 as it moves to the limiting external membrane surface of sarcolemma and transverse-tubules in response to insulin, contraction or activators of energy-status signalling, including hypoxia. HA-GLUT4-GFP is largely excluded from the sarcolemma and transverse-tubule membrane of cardiomyocytes under basal conditions, but is similarly trafficked to these membrane surfaces after stimulation with insulin, contraction or hypoxia. Internalisation of sarcolemma GLUT4 has been investigated by pulse-labelling surface GLUT4 with intact anti-HA antibody. At early stages of internalisation, HA-tagged GLUT4 colocalises with clathrin at puncta at the sarcolemma, indicating that in cells returning to a basal state, GLUT4 is removed from external membranes by a clathrin-mediated route. We also observed colocalisation of GLUT4 with clathrin under basal conditions. At later stages of internalisation and at steady state, anti-HA antibody labeled-GLUT4 originating from the sarcolemma was predominantly detected in a peri-nuclear compartment, indistinguishable among the specific initial stimuli. These results taken together imply a common pathway for internalisation of GLUT4, independent of the initial stimulus.